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Plants as Expression System for Recombinant Therapeutic Glycoproteins

INTERNATIONAL SCIENTIFIC CONFERENCE “EARTH BIORESOURCES AND ENVIRONMENTAL BIOSAFETY: CHALLENGES AND OPPORTUNITIES” Kyiv, Ukraine, November 4-7, 2013. Plants as Expression System for Recombinant Therapeutic Glycoproteins. Herta Steinkellner, Richard Strasser and Josef Glössl.

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Plants as Expression System for Recombinant Therapeutic Glycoproteins

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  1. INTERNATIONAL SCIENTIFIC CONFERENCE “EARTH BIORESOURCES AND ENVIRONMENTAL BIOSAFETY: CHALLENGES AND OPPORTUNITIES” Kyiv, Ukraine, November 4-7, 2013 Plants as Expression System for Recombinant Therapeutic Glycoproteins Herta Steinkellner, Richard Strasser and Josef Glössl University of Natural Resources and Life Sciences, Vienna, Austria Department of Applied Genetics and Cell Biology www.boku.ac.at

  2. Glycoproteins Glycosylation of proteins • N-Glycosylation: Asn-linked glycosylation (Asn-X-Ser/Thr) N-glycans • O-Glycosylation: Ser/Thr-linked glycosylation O-glycans Glycosphingolipids P. Stanley, 2011 Cold Spring Harbor Laboratory Press

  3. N-glycosylation Function of N-glycans: - Protein folding and stability - Protein targeting (e.g. for lysosomal enzymes) - Protein-protein or protein-carbohydrate interactions (lectins) - Biological activity of proteins (e.g. effector functions of IgGs) - Control of protein half-life (e.g. erythropoietin)

  4. In CHO expression system: A mixture of glycoforms Why glyco-engineering ? • Changes in N-glycan structure may significantly affect protein confirmation and pharmacokinetic behaviour, thus influencing e.g. antigen binding and effector functions in case of monoclonal antibodies • Current expression systems generate a mixture of glycoforms • Substantial deficits exist in understanding the role of specific glycosylation patterns on therapeutic proteins like monoclonal antibodies • Well defined glycoforms on recombinant proteins are urgently needed • Expression systems are needed which produce “tailor-made” N-glycan structures

  5. General goals: Production of recombinant glycoproteins with a defined, homogeneous glycosylation profile in order to study the impact of glycosylation the therapeutic potency of various glycoforms Further development of plant expression systems for therapeutically relevant glycoproteins: Why glyco-engineering ?

  6. Plant expression systems are rapidly evolving as expression systems for therapeutically relevant proteins, since they are convenient, biologically safe cost-effective However, since plants differ in certain aspects of their N-glycosylation pathway from that in human, “humanization” of the N-glycan biosynthetic pathway is needed in order to avoid immunological problems get authentic N-glycan structures Why glyco-engineering in plants?

  7. N-glycosylation in mammalian cells • Prominent example: • monoclonal antibodies (mAbs): • IgG1 heavy chain has conserved N-glycosylation site (Asn 297) •  N-glycan structure influences biological activity of antibodies

  8. Schematic presentation of the N-glycosylation pathways in humans, yeast, insect cells and plants The common ER-resident oligosaccharide precursor acts as starting point for further modifications along the Golgi apparatus Legend:

  9. ManI GnTI ManII GnTII N-Glycan Processing in Plantsvs.Mammals cis Golgi trans Golgi medial Golgi 1,6-FucT Mammals 2,6-SiaT b1,4-GalT 1,3-FucT Xylose N-acetylglucosamine Plants Galactose Mannose Fucose b1,2-XylT Sialic acid b1,3-GalT α1,4-FucT Plant and mammalian N-glycan processing steps differ in the late Golgi steps

  10. CMP Engineering of the N-Glycan Processing Pathway in Plants Goal: „Humanised“ N-glycan structures Plant-type N-glycan Strategy: • by • knock out (A. thaliana) • RNAi (N. benthamiana) • Removalof • β1,2-xylosyltransferase • core α1,3-fucosyltransferase Transformation with β1,4-galactosyltransferase • Engineering of the sialic acid pathway by: • Transformation of six genes required for the biosynthesis of CMP-Neu5Ac and transport into the Golgi lumen • Transformation with α2,6-sialyltransferase „Humanized“ N-glycan

  11. Reconstruction of the human sialylation pathway in plants The genes for 6 proteinshadtobeintroducedinto plant cell, permittingthebiosynthesisofsialicacid (Neu5Ac) , itsactivation, transportintothe Golgi, andtransferonto terminal galactose A. Castilho et al., J. Biol. Chem.285, 15923, 2010

  12. 2 Examples of mAbs against viruses: anti HIV antibdy 2G12: (Polymun, Dept. Biotechnology, BOKU, Vienna) anti EBOLA virus antibody 13F6 (Mapp Biopharmaceutical, CA, USA) Different functional activities of monoclonal antibody (mAb) N-glycoforms

  13. Surface plasmon resonance AA GnGn sFcgRIII CHO GnGnF6 GnGnXF3 Fc from: Sondemann et al., 2000 Functional activities of mAbs glycoforms mAb 2G12 bindingtoFcγreceptorIIIa (CD16a) FucosedeficientmAb 2G12 exhibitshigherbindingto CD16  Higher anti HIV activityofmAb 2G12 D.N. Forthal et al., J. Immunol. 185, 6876, 2010

  14. GnGn CHO GnGnXF Functional activities of mAbs glycoforms mAb 13F6 (anti Ebola virus) Virus protection assay in mice Kevin Whaley

  15. Summary and Outlook • It is now possible to produce complex human proteins for therapeutic purposes, largely correctly folded and N-glycosylated, in plants • Plants have demonstrated a high degree of tolerance to changes in the N-glycosylation pathway, allowing recombinant proteins to be modified into human-like structures • Glyco-engineering haspavedthewaytofullyhumanizethe plant N-glycosylationpathway • Frequently the results are a largely homogeneously glycosylated protein, enabling to study • the impact of glycosylation • the therapeutic potency of various glycoforms • There is increasing evidence for the significance of proper N-glycosylation for the efficacy of biopharmaceuticals • Glyco-engineering has become an important issue not only for academia but also for the biopharmaceutical industry.

  16. Acknowledgements BOKU - University of Natural Resources and Life Sciences, Vienna Department of Applied Genetics and Cell Biology Herta Steinkellner Richard Strasser Alexandra Castilho Pia Gattinger Jakub Jez Department of Chemistry (N-glcosylation analyses) Friedrich Altmann Johannes Stadlmann Department of Biotechnology (HIV testing) Renate Kunert Heribert Quendler Supported by: Austrian Science Fund European Commission

  17. University of Natural Resources and Life Sciences, Vienna (BOKU)congratulates to the 115th Anniversary of NULES of Ukraine and the 15th Anniversary of GCHERA Prof. Dr. Josef Glössl Vice Rector for Research and International Research Collaboration josef.gloessl@boku.ac.at www.boku.ac.at 17

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